Abstract

Human microsomal epoxide hydrolase (mEH), encoded by the
EPHX1 gene, is involved in the metabolism of tobacco
carcinogens. We investigated the effect of exon 3 and 4 polymorphisms
of the EPHX1 gene in 121 patients with cancers of the
oral cavity/pharynx, 129 patients with cancer of the larynx, and 172
non-cancer controls, all Caucasian regular smokers. The potential
modifying role of previously analyzed GSTM1,
GSTM3, and GSTP1 genotypes was also
examined. Compared with the putative low-activity genotypes, odds
ratios (ORs) associated with predicted intermediate and high mEH
activity genotypes were significantly increased for oropharyngeal
cancers [OR = 1.8; 95% confidence interval
(CI) = 1.0–3.3; and OR = 2.1; 95%
CI = 1.0–4.5, respectively;
Ptrend = 0.03] and laryngeal
cancers (OR = 1.7; 95% CI = 1.0–3.1;
and OR = 2.4; 95% CI = 1.1–5.1,
respectively; Ptrend = 0.02).
Moreover, a positive interaction was found between mEH activity and
GSTM3 genotype for laryngeal cancer. The combined
EPHX1 high activity-associated genotype and
GSTM3 (AB or BB) genotype
conferred a 13.1-fold risk (95% CI = 3.5–48.4) compared
with the concurrent presence of the EPHX1 low
activity-associated genotype and the GSTM3 AA genotype.
Thus, EPHX1 polymorphisms may be one of the factors of
importance in susceptibility to smoking-related cancers of the upper
aerodigestive tract.

Introduction

France is among the countries with the highest incidences of
cancers of the upper aerodigestive tract in men
(1)
.
Tobacco smoking and alcohol consumption are the main risk factors for
these malignancies
(2)
. However, the risk of developing
smoking-related cancers varies between individuals, and there is
increasing evidence that polymorphisms in the genes that encode enzymes
involved in the metabolism of tobacco carcinogens could modify this
risk
(3)
.
mEH,
3
encoded by the EPHX1 gene, catalyzes the hydrolysis of
reactive epoxide intermediates
(3)
. This reaction usually
is regarded as a detoxifying pathway because the metabolites produced
are less reactive and can be more easily excreted. mEH also intervenes
in the metabolic activation of the polycyclic aromatic hydrocarbons
abundant in tobacco smoke, thereby triggering the formation of highly
reactive metabolites
(4)
. Two variant EPHX1
alleles have been associated with altered mEH activity in Caucasians;
substitution of histidine for tyrosine at residue 113 (exon 3
polymorphism) decreases mEH activity, whereas substitution of arginine
for histidine at residue 139 (exon 4 polymorphism) enhances enzyme
activity, probably by affecting the stability of the mEH protein. When
both mutations are present, mEH activity approximately equals normal
(5)
. The mEH enzyme is expressed in the upper
aerodigestive tract
(6)
, which makes it an interesting
candidate as a modifier of smoking-associated disorders at this site.
However, few studies have investigated the role of mEH in the onset of
several smoking-associated cancers, and the results of these studies
were inconclusive
(7, 8)
We recently reported an almost
3-fold risk of lung cancer associated with predicted high mEH activity
based on data obtained in a multicentric hospital-based case-control
study
(9)
. We investigated in this study the relationships
between EPHX1 polymorphisms and cancers of the upper
aerodigestive tract and whether the effects of these polymorphisms were
modified by GSTM1, GSTM3, and GSTP1
genotypes.

Materials and Methods

Details of the study have been described previously
(10)
. In brief, Caucasian individuals were recruited
between 1988 and 1992 in 10 French hospitals. Peripheral blood samples
were available from 121 patients with cancers of the oral
cavity/pharynx (67 oral cancers, 50 oro- or hypopharyngeal cancers, and
4 unspecified or unclassifiable cancers of the oral cavity and
pharynx), 129 patients with cancers of the larynx (55 supraglottic, 47
glottic/subglottic, and 27 unspecified or unclassifiable larynx
cancers), and 172 control individuals. Only incident cases with
histologically confirmed primary squamous cell carcinoma were included.
The control group was frequency matched on age, sex, and hospital. The
main diagnoses among control individuals were rheumatological (33%),
infectious and parasitic diseases (10%), respiratory (9%),
cardiovascular (8%), digestive diseases (6%), and traumatological
diseases (6%). The main reasons for admission were related to general
symptoms (7%) for the other categories. Severe liver diseases (total
bilirubin >60 μmol/l, or serum glutamic-oxaloacetic transaminase or
glutamic-pyruvic transaminase >150 units/l, or serum phosphatase
alkaline >600 units/ml) were exclusion criteria for both cases and
control subjects. All study subjects were regular smokers, defined as
people having smoked at least five cigarettes (or cigars or pipes) per
day for at least 5 years. Detailed information on recent and past
tobacco use and alcohol consumption was recorded during a personal
standardized interview. The daily consumption of each type of tobacco
was expressed in g/day (1 g for cigarette, 2 g for cigar, and
3 g for pipe). The average daily consumption of tobacco smoking
was calculated by dividing the cumulative lifetime tobacco consumption
by the overall duration of smoking. The consumption of alcoholic
beverages was expressed in grams of pure ethanol (4.0, 9.4, 14.5, and
31.7 g for 0.1 liter of beer, wine, cider, aperitif, and hard
liquor, respectively). The average daily consumption of alcohol was
calculated by dividing the cumulative daily consumption of alcohol (the
sum of the number of grams of ethanol per day multiplied by the number
of years that the quantity was drunk) by the overall duration of
drinking
(11)
. The main characteristics of the study
population are presented in Table 1
⇓
.

Blood samples were collected into EDTA tubes and stored at −20°C
until total WBC DNA was extracted, using standard protocols. The
EPHX1 genotyping assay was performed blinded to the
subjects’ case-control status. After two separate PCR reactions, the
variant allele correlating with increased mEH activity
(Arg139) was determined by the presence of a RsaI
restriction site
(6)
, and the allele correlating with
decreased activity (His113) was determined by the presence
of an AspI site
(12)
. Three levels of predicted
mEH activity were assigned according to in vitro expression
of the variant alleles
(8, 9)
. GSTM1,
GSTM3, and GSTP1 genotypes had been determined
previously as described
(11)
. ORs and their 95% CIs were
calculated by unconditional logistic regression, with sex, age (<50,
50–54, 55–59, 60–64, and ≥65 years), daily consumption of tobacco
in g/day (20, 21–30, and ≥31), duration of smoking in years (25,
26–34, and ≥35), exclusive cigarette smoking (no/yes), and daily
consumption of alcohol in g/day (40, 41–80, 81–120, and ≥121) as
confounding factors. All of the cutoff points were defined according to
the distributions in the control population so that sufficient numbers
of individuals were included in each subgroup. We evaluated the
gene-dosage effect (i.e., increasing risks according to
predicted intermediate and high mEH activity compared with predicted
low activity) by linear trend tests
(13)
. All P
values reported are two-sided.

Results and Discussion

In the control population, exon 3 and 4 genotype frequencies were
in Hardy-Weinberg equilibrium (P = 0.65 and
0.48, respectively; Table 2
⇓
), and were similar across disease groups. Significant differences in
the distribution of exon 3 genotypes were found between controls and
both oral/pharynx cancers (P = 0.004) and
larynx cancers (P = 0.006), with a
significant gene-dose effect for larynx cancer
(Ptrend = 0.006) and to a
less extent for oral/pharynx cancer
(Ptrend = 0.17). In
contrast, exon 4 genotype distributions were similar in cancer cases
and controls.

Distribution of individuals and ORs of cancer according to EPHX1
genotypes and predicted mEH activity

The distribution of the predicted mEH activity was significantly
different between control subjects and oral/pharynx cancer patients
(P = 0.03) or larynx cancer patients
(P = 0.01; Table 2
⇓
). A significant increase
in risk with increasing mEH activity was found for oral/pharynx cancer
(Ptrend = 0.03) and larynx
cancer (Ptrend = 0.02); in
particular, individuals with predicted high activity were at a >2-fold
risk of developing these cancers (OR = 2.1; 95%
CI = 1.0–4.5 for oral/pharyngeal cancer; and
OR = 2.4; 95% CI = 1.1–5.1 for
laryngeal cancer). Overall, the present findings are consistent with
our previous observations on lung cancer
(9)
. The ORs
associated with predicted mEH activity were not modified by the
duration of smoking (dichotomized at the approximate median in the
control population), combined smoking and alcohol exposures (>20 g
tobacco/day, and >80 g ethanol/day versus others),
GSTM1 genotype, or GSTP1 genotype (data not
shown). In contrast, we found a significant interactive effect between
predicted mEH activity and GSTM3 genotype on larynx cancer
risk (Table 3)
⇓
. Carriers of both the combined EPHX1 high
activity-associated genotype and the GSTM3 (AB or
BB) genotype had a 13.1-fold risk (5% CI = 3.5–48.4) compared with individuals with the concurrent presence of
the EPHX1 low activity-associated genotype and the
GSTM3 AA genotype. When we used low mEH activity as the
reference category, a significant increase in risk associated with high
mEH activity was observed in carriers of the GSTM3
(AB or BB) genotype (OR = 6.4;
95% CI = 1.5–27.3), but not in carriers of the
GSTM3 AA genotype (OR = 1.1; 95%
CI = 0.4–3.1). A similar tendency was shown for
oral/pharyngeal cancers, but the interaction test did not reach
statistical significance (Table 3)
⇓
. These findings, however, were based
on very small numbers and should be confirmed in larger studies.

Number of cases/controls and ORsa
of cancer according to
predicted mEH activity and GSTM3 genotype

A potential limitation of our study would be the use of hospital
controls, especially if there are any associations between
EPHX1 genotypes and diseases diagnosed in the control group.
Nevertheless, no statistically significant association was found within
this control group between genotypes and the main diseases diagnosed,
although the likelihood of finding a difference in genotype
distribution is low. Moreover, if increased mEH activity was associated
with smoking-related diseases among controls, this would result in
underestimation of the real relative risks. Exclusion of the 28
controls with pulmonary or cardiovascular diseases did not modify the
cancer risks associated with mEH activity. In addition, the frequencies
of EPHX1 genotypes reported in our study are comparable to
those observed in other Caucasian populations
(12)
. Taken
together, this study suggests that EPHX1 genotypes
associated with high mEH activity are associated with increased risk of
smoking-related cancers of the oral cavity, pharynx, and larynx,
confirming our prior results on lung cancer
(9)
. The
observed 2-fold risk of upper aerodigestive tract cancers associated
with predicted mEH activity is what we should expect for a
low-penetrance susceptibility gene. However, the implications for
public health could be very important given the widespread prevalence
of alleles associated with high mEH activity in Caucasian populations.
Thus, EPHX1 polymorphism may be a significant genetic
determinant of smoking-induced cancers.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.